Effects Of Troxerutin On Gene Expression In Mice

Troxerutin(TX),is a synthetic derivative of rutin which is a natural bioflavonoid extracted from Styphnolobium japonicum(L.)Schott.It is abundant in olive,buckwheat,asparagus,coffee,and grain etc.It has anti-inflammatory immune regulation,anti-allergy,antiulcer,liver and kidney dirty protection,and many other pharmacological effects.It dissoves in water,and is easily absorbed.It can effectively reduce capillary brittleness and abnormal leakage,prevent the formation of thrombus,improve vascular injury,have a significant protective effect on acute cerebral ischemic injuries.In the past few decades,although it had a lot of experiments to explore different aspects of TX’s pharmacological activities,the effects o f TX on genome-wide expression have not been reported,which could help us understanding the clinical effects of TX.Purpose:In this context,we used Illumina HiSeq 4000 to generate a substantial dataset of transcript reads in different groups treated with or without TX.Using NCBI,Uniprot,GO database to annotate differentially expressed genes.And to further explain how TX involved in biological process,and find its plays in the process of active related genes and signaling pathways.Methods:1.The mice of 5 weeks were divided into the experimental groups and the control groups.The control groups received injection of normal saline(NS),and the experimental group received subcutaneous injection of TX dissolved in NS at dose of 130 μg/g twice daily at 9:00 AM and 5:00 PM.On the 3th day of treatment,the blood samples were drawn from the mice’ hearts and the library was constructed;2.We used the Anon Illumina 4000 sequencing platform to carry on transcriptional sequencing analysis of samples,and sorting,filtering and quality assessment of the raw data;3.Using the existing genome-wide database,the sequencing results were compared with the reference genome,and then calculated the expression quantity;4.Using DESeq to carry out the analysis of different genes expression,compared with the treatment group and reference group,we selected standard was |log2Ratio|>1 and q<0.05,then got up-regulated and down-regulated genes,and the differentially expressed genes were annotated with NCBI,Uniprot,GO;5.Q-PCR verified the sequencing results.Results:1.Based on blood transcriptome sequencing analysis of 6 samples,we got the raw reads 149,144,776,and the clean reads 140,324,838,it account for 90%of total data.Compared with the reference genome,the results showed that over 88.5%of the data were mapped to the reference genome for each sample and the Q30 score was greater than 95%.For the control group and experiment group,95.35%and 96.04%were mapping to the exon region,3.12%and 2.84%belonged to the intron region,and 1.53%and 1.12%were in the intergenic sequence respectively;2.Transcriptomic analysis demonstrated that the expression of fifteen genes was significantly changed by the treatment with TX,among which 5 genes were up-regulated and 10 genes were down-regulated.DAG analysis showed that these genes could influence T cell-mediated cytotoxicity(q=0.000274),telencephalon development(q=0.000374),cell surface(q=5.04e-06),endoplasmic reticulum exports(q=0.000153),β2-microglobule binding(q=0.000278),TAP-binding(q=0.000165),and T cell and neuropeptide Y receptor binding(q=0.000566),which several entries were significantly enriched.According to these results,we speculated that it might be related to the TX’s anti-inflammation immunity and nerve protection;3.The Q-PCR results were consistent with sequencing.Conclusion:In this study,we made a comprehensive analysis of the effects of TX on genome-wide expression of genes.According to this experiment,we found out the differentially expressed genes behind the TX treatment with mice,and explored the signal pathways which gene involved and discussed the connection between the gene and TX.In general,TX can regulate the expression of these genes levels to participate in the anti-inflammatory immune,brain development,cardiovascular physiology etc formation process,to play a protective role.